The present invention relates to compounds and pharmaceutically acceptable salts thereof and methods for treating infections by HIV and related viruses and/or the treatment of Acquired Immune Deficiency Syndrome (AIDS) Also disclosed are pharmaceutical compositions containing the compounds and the method of use of the present compounds alone or in combination with other agents, for the treatment and inhibition of ATDS and viral infection from HIV.
A retrovirus designated Human Immunodeficiency Virus (HIV) is believed to be the causative agent of the complex disease termed Acquired Immune Deficiency Syndrome (AIDS) and is a member of the lentivirus family of retroviruses (M. A. Gonda, F. Wong-Staal N R. C. Gallo, xe2x80x9cSequence Homology and Morphological Similarity of HTLV III and Visna Virus, A Pathogenic Lentivirusxe2x80x9d, Science, 227, 173, (1985); and P. Sonigo and N. Alizon, et al., xe2x80x9cNucleotide Sequence of the Visna Lentivirus: Relationship to the AIDS Virusxe2x80x9d, Cell, 42, 369, (1985)). The HIV virus (also referred to as the AIDS virus) was previously known as or referred to as LAV, HTLV-III, or ARV, and is now designated by HIV-1. Other closely related variants of HIV-1 include HIV-2 and SIV (simian immunodeficiency virus), and mutants thereof. The complex disease AIDS includes progressive destruction of the immune system and degeneration of the central and peripheral nervous system. The HIV virus appears to preferentially attack helper T-cells (T-lymphocytes or OKT4-bearing T-cells) and also other human cells, e.g., certain cells within the brain. The helper T-cells are invaded by the virus and the T-cell becomes an HIV virus producer. The helper T-cells are quickly destroyed and their number in the human being is depleted to such an extent that the body""s B-cells as well as other T-cells normally stimulated by helper T-cells no longer function normally or produce sufficient lymphokines and antibodies to destroy the invading virus or other invading microbes.
While the HIV virus does not necessarily cause death per se, it does cause the human""s immune system to be so severely depressed that the human falls prey to various other diseases such as herpes, Pneumocystis carinii, toxoplasmosis, cytomegalovirus, Kaposi""s sarcoma, and Epstein-Barr virus related lymphomas among others. These secondary infections are separately treated using other medications as is conventional. Early during infection, humans with HIV virus seem to live on with little or no symptoms, but have persistent infections. Later in the disease, humans suffer mild immune system depression with various symptoms such as weight loss, malaise, fever, and swollen lymph nodes. These syndromes have been called persistent generalized lymphadenopathy syndrome (PGL) and AIDS related complex (ARC) and develop into AIDS.
In all cases, those infected with the AIDS virus are believed to be persistently infective to others. Further, AIDS and AIDS related complex is after some time fatal.
A description of the mechanism by which the virus infects its host is given in an article by R. Yarchoan, and S. Broder, xe2x80x9cDevelopment of Antiretroviral Therapy for the Acquired Immunodeficiency Syndrome and Related Disordersxe2x80x9d, New England Journal of Medicine, 316, 557-564 (Feb. 26, 1987).
Considerable efforts are being directed toward the control of HIV by means of inhibition of the reverse transcriptase of HIV, required for replication of thee virus. (V. Merluzzi et al., xe2x80x9cInhibition of the HIV-1 Replication by a Nonnucleoside Reverse Transcriptase Inhibitorxe2x80x9d, Science, 25, 1411 (1990)). For example, a currently used therapeutic compound, AZT, is an inhibitor of the viral reverse transcriptase (U.S. Pat. No. 4,724,232). Unfortunately, many of the now used compounds suffer from toxicity problems, lack of bioavailability or are short lived in vivo, viral resistance, or combinations thereof. Therefore, new compounds are being investigated. For example, a PCT application published on Feb. 18, 1993, (WO 93/03022; International Application No. PCT/SE92/00533) discloses thiourea compounds which show promise in the treatment and inhibition of HIV and AIDS.
Certain compounds falling within the scope of the definition of the methods of the present invention have been published in roles other than for the treatment of HIV. These compounds are excluded by proviso from the compound claims. The Chemical Abstract registry numbers or sources for these compounds are as follows:
Therefore, it is an object of the invention to provide compounds and pharmaceutically acceptable salts thereof to inhibit and/or treat HIV and AIDS.
Another object of the present invention is to provide therapeutic formulations that are of value in the inhibition and/or treatment of infection by HIV and the treatment or inhibition of the acquired immune deficiency syndrome.
Another object is to provide methods for the inhibition and/or treatment of infection by HIV and the resulting acquired immune deficiency syndrome.
Other objects, features, and advantages will become apparent to those skilled in the art from the following description and claims.
The present invention provides compounds useful for the inhibition and/or treatment of HIV and AIDS, either as compounds, pharmaceutically acceptable salts, pharmaceutical composition Ingredients, whether or not in combination with other anti-virals, immunomodulators, antibiotics, or vaccines. Methods of treating or inhibiting AIDS, methods of inhibiting replication of HIV, and methods of treating or inhibiting HIV in humans are also disclosed.
The compounds used in the methods of the present invention are those of the formula (IA) below 
wherein A is 
wherein Ra is H, ORb, CN, NO2, N(Rb)2, SRb, SO2Rb, SO2N(Rb)2, CORb, CO2Rb, CON(Rb)2, PO(Rb)2, PO(ORb)2, PO(NRb)2, wherein Rb is hydrogen, C1-C6 alkyl, C1-C6 substituted alkyl, C2-C6 alkenyl, C2-C6 substituted alkenyl, C2-C8 alkynyl, C2-C8 substituted alkynyl, C1-C6 alkoxy, C1-C6 substituted alkoxy, C4-10 aralkyl, C1-10 alkaryl, C1-10 alkylthio, C4-10 aralkylthio, C1-10 alkylsulfinyl, C4-10 alkylsulfinyl, C1-10 alkylsulfonyl, C4-10 alkylsulfonyl, carboxy, C1-10 alkylthiocarbonyl, C4-10 aralkylcarbonyl, C4-10 aralkylthiocarbonyl, C4-10 aralkoxycarbonyl, C4-10 aralkoxycarbonyl, C1-4 alkyl, C4-10 aralkoxy, C1-12 dialkylamino-C1-6 aralkanoylamino C4-10 aralkylamino or C1-C4 alkanoyloxy;
R1 is a stable saturated or unsaturated, substituted or unsubstituted, 3 to 8 membered organic monocyclic ring having 0 to 4 hetero atoms selected from S, O, and N; or R1 is a stable, saturated or unsaturated, substituted or unsubstituted, 7 to 10 membered organic bicyclic ring having 0 to 5 hetero atoms selected from S, O, and N;
R2 is a group of the formula 
xe2x80x83wherein R5 is R1 as defined above; or R5 is a group of the formula
(R10)yxe2x80x94Xxe2x80x94
xe2x80x83wherein y is 1 or 2; X is N, S, O and R10 is R1 as defined; or R10 is hydrogen, C1-C6alkyl, C2-C6 alkenyl, or C2-C6 alkynyl, substituted C1-C6alkyl, substituted C2-C6 alkenyl, or substituted C2-C6 alkynyl; or R5 is hydrogen, halo, cyano, carboxy, amino, thio, hydroxy, C1-C6 alkoxy, C1-C6 substituted alkoxy, C1-C6 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, or C2-C8 alkenoxy;
R6, R7, R8, and R9 are independently C3-C8 cycloalkyl, hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted C1-C6alkyl, substituted C2-C6 alkenyl, or substituted C2-C6 alkynyl, C1-C6 substituted alkoxy, halo, amino, nitro, cyano, C1-C5 alkoxy, hydroxy, carboxy, hydroxymethyl, aminomethyl, carboxymethyl, C1-C4 alkylthio, C1-C4 alkanoyloxy, carbamoyl, or a halo substituted C1-C6 alkyl; or two of which, along with the carbons to which they are attached, combine to form a stable, saturated or unsaturated, substituted or unsubstituted, 3 to 7 membered organic monocylic ring having 0 to 4 hetero atoms selected from S, O, or N; or R6 and R8, or R7 and R9, along with the carbon to which they are attached, form a stable, saturated or unsaturated, substituted or unsubstituted, 3 to 7 membered organic monocylic ring having 0 to 4 hetero atoms selected from S, O, or N;
R3 and R4 are independently hydrogen, hydroxy, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted C1-C6alkyl, substituted C2-C6 alkenyl, or substituted C2-C6 alkynyl, substituted alkoxy, amino, cyano, nitro, C1-C6 alkoxy, C1-C6 substituted alkoxy, carboxy, hydroxymethyl, aminomethyl, carboxymethyl, C1-C4 alkylthio, C1-C4 alkanoyloxy, halo-substituted (C1-C6)alkyl, or carbamoyl; or salts thereof;
It should also be understood that when the term xe2x80x9cHIVxe2x80x9d is used, it includes HIV-1, components, mutant variations, subtypes, and serotypes thereof, and related viruses, components, mutant variations, subtypes, and serotypes thereof. When the term xe2x80x9cinhibitxe2x80x9d is used, its ordinary meaning is intended, which is to prohibit, hold in check, or discourage, and is not to be construed to be limited to a particular process, procedure, or mechanism of action.
The terms xe2x80x9cstable, saturated or unsaturated, substituted or unsubstituted, 3 to 8 memberedxe2x80x9d, or xe2x80x9c3 to 7 membered organic monocyclic ring having 0 to 4 hetero atoms selected from S, O, and Nxe2x80x9d include those wherein the nitrogen and sulfur hetero atoms are optionally oxidized, and the nitrogen hetero atom optionally quaternized. The substituted ring may have 1-8 substituents independently selected from aryl, substituted aryl, halo, C1-C6 alkyl, C1-C5 alkoxy, C1-C6 alkenyl, C2-C8 alkynyl, C2-C8 alkenoxy, amino, nitro, cyano, carboxy, hydroxymethyl, aminomethyl, carboxymethyl, C1-C4 alkylthio, hydroxy, C1-C4 alkanoyloxy, carbamoyl, halo-substituted C1-C6 alkyl, C1-C6 alkoxy-substituted C1-C6 alkyl, a group of the formula
xe2x80x94SO2Rx
wherein Rx is C1-C6 alkyl, aryl, substituted aryl, or amino; or a group of the formula 
wherein Rx is as defined above.
The term xe2x80x9cstable, saturated or unsaturated, substituted or unsubstituted, 7 to 10 membered organic bicyclic rings having 0 to 5 hetero atoms selected from S, O, and Nxe2x80x9d includes those wherein the nitrogen and sulfur hetero atoms are optionally oxidized, and the nitrogen hetero atom(s) optionally quaternized. The bicyclic rings may be substituted 1 to 8 times, the substituents independently selected from those above listed for the monocyclic rings.
Examples of such monocyclic and bicyclic rings are cyclo(C3-C8)alkyl, cyclo(C3-C8)alkenyl; isothiazolyl, substituted isothiazolyl, tetrazolyl, substituted tetrazolyl, triazolyl, substituted triazolyl, pyridyl, substituted pyridyl, imidazolyl, substituted imidazolyl, phenyl, substituted phenyl, naphthyl, substituted naphthyl, benzoxazolyl, substituted benzoxazolyl, benzimidazolyl, substituted benzimidazolyl,thiazolyl, substituted thiazolyl, oxazolyl, substituted oxazolyl, benzothiazolyl, substituted benzothiazolyl, pyrazinyl, substituted pyrazinyl, pyridazinyl, substituted pyridazinyl, thiadiazolyl, substituted thiadiazolyl, benzotriazolyl, substituted benzotriazolyl, pyrrolyl, substituted pyrrolyl, indolyl, substituted indolyl, benzothienyl, substituted benzothienyl, thienyl, substituted thienyl, benzofuryl, substituted benzofuryl, furyl, substituted furyl, quinolinyl, substituted quinolinyl, isoquinolinyl, substituted isoquinolinyl, pyrazolyl, and substituted pyrazolyl. Other examples of such ring systems may be found in J. Fletcher, O. Dermer, R. Fox, Nomenclature of Organic Compounds, pp. 20-63 (1974), and in the Examples herein.
The term xe2x80x9cC1-C6 alkylxe2x80x9d includes such groups as methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl, t-butyl, n-pentyl, n-hexyl, 3-methylpentyl, and the like.
The term xe2x80x9chaloxe2x80x9d and xe2x80x9chalogenxe2x80x9d refer to chloro, bromo, fluoro, and iodo.
xe2x80x9cC1-C6 alkoxyxe2x80x9d refers to those groups such as methoxy, ethoxy, propoxy, t-butoxy, and the like.
xe2x80x9cC2-C6 alkenylxe2x80x9d refers to those groups such as vinyl, 1-propene-2-yl, 1-butene-4-yl, 1-pentene-5-yl, 1-butene-1-yl, and the like.
xe2x80x9cC1-C10 alkylthioxe2x80x9d refers to those groups such as methylthio, ethylthio, t-butylthio, and the like.
xe2x80x9cC1-C4 alkanoyloxyxe2x80x9d refers to those groups such as acetoxy, propionoxy, formyloxy, butyryloxy, and the like.
The term xe2x80x9cC2-C8 alkenoxyxe2x80x9d includes groups such as ethenyloxy, propenyloxy, iso-butoxy ethenyl, and the like.
The term xe2x80x9cC2-C8 alkynylxe2x80x9d includes groups such as ethynyl, propynyl, pentynyl, butynyl, and the like.
The term halo-substituted C1-C6 alkyl includes alkyls substituted 1, 2, or 3 times by a halogen, including groups such as trifluoromethyl, 2-dichloroethyl, 3,3-difluoropropyl, and the like.
The term xe2x80x9caminexe2x80x9d includes groups such as NH2, NHCH2 and N(CH)2 and the like which may be optionally substituted with halogen, amino, C1-C7 acyloxy, C1-C6 alkyl, C1-C6 alkoxy, nitro, carboxy, carbanoyl, carbanoyloxy, cyano or methylsulphonylamino and the like.
The terms xe2x80x9ccarboxylxe2x80x9d, xe2x80x9ccarboxymethylxe2x80x9d and xe2x80x9ccarbamoylxe2x80x9d include the corresponding pharmaceutically acceptable C1-C6 alkyl and aryl esters.
The term xe2x80x9carylxe2x80x9d includes 3 to 8 membered stable saturated or unsaturated organic monocyclic rings having 0 to 4 hetero atoms selected from S, O, and N; and 7 to 10 membered organic stable, saturated or unsaturated, bicyclic rings having 0 to 5 hetero atoms selected from S, O, N; both of which may be substituted by halo, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, substituted C1-C6alkyl, C1-C6 substituted alkoxy, substituted C2-C6 alkenyl, or substituted C2-C6 alkynyl, substituted alkoxy, amino, nitro, cyano, carboxy, hydroxymethyl, aminomethyl, carboxymethyl, C1-C4 alkylthio, hydroxy, C1-C4 alkanoyloxy, carbamoyl, or halo-substituted C1-C6 alkyl.
The terms substituted alkyl, substituted alkenyl, substituted alkynyl and substituted alkoxy are these substitutents substituted with halogen, hydroxy, amino, C1-C7 acyloxy, nitro, carboxy, carbanoyl, carbanoyloxy, cyano, methylsulfonylamino, or C1-C6 alkoxy, and may be substituted once or twice with the same or different group.
Preferred definitions are when R3 and R4 are hydrogen; and
R1 is a heteroaromatic monocyclic or bicyclic ring as defined above and has the Nxe2x80x2-linkage at the 3 position, or more preferably at the 2 position, relative to a hetero atom
Examples of R1 are thiazolyl, (4-methyl)thiazolyl, (4,5-dimethyl)thiazolyl, (4-cyano)thiazolyl, (4-trifluoromethyl)thiazolyl, benzothiazolyl, (6-fluoro)benzothiazolyl, (6-chloro)pyraziny, (4-ethyl) thiazolyl, 4-(3-pyridyl)thiazolyl, 4-(3-nitrophenyl)thiazolyl, pyridyl, (6-bromo)pyridyl, (6-chloro)pyridyl, (6-methyl)pyridyl, (5-methyl)pyridyl, (6-trifluoromethyl)pyridyl, (5-trifluoromethyl)pyridyl, (6-ethyl)pyridyl, (5-ethyl)pyridyl, (6-bromo)pyrazinyl, 3-(6-bromo)pyridazinyl, (5-cyano)pyridyl, (5-cyano)pyridyl, (5-cyano)pyrazinyl, (6-cyano)pyrazinyl, 3-(6-cyano)pyridazinyl, 1,3,4-thiadiazoyl, benzimidazolyl, imidazolyl, (5-bromo)pyridyl, (5-chloro)pyridyl, (5-chloro)pyrazinyl, (5-bromo)pyrazinyl, (6-chloro)pyridazinyl, 2-(3-[6-bromo]pyridazinyl), 2-(3-[6-chloro]pyridazinyl) 2-(3-[6-cyano]pyridazinyl), 2-(3-ethyl)pyridyl, 3-(6-methoxy)pyridazinyl, 2-(5-nitro)pyridyl, 2-(5,6-dichloro-4-azabenzimidazolyl), 4-(6-aminopyrimidinyl), 4-pyrimidinyl, 2-(3-pyridazlnyl), 2-(3-(6-methyl)pyridazinyl, 2-pyrazinyl, 2-(5-methyl)pyrazinyl); and R2 are (4-methyl) -3-pentenyl, (xc2x1)-cis-N-(3,4-benzo-cis-bicyclo-[3.1.0]-hexen-6-yl), 
The preferred optional substituents for R1 comprise mono, di, or tri halo, preferably bromo or chloro, especially para to the Nxe2x80x2-linkage.
The preferred optional substituents to R5 are halo or C1-C6 alkoxy, especially 2,6-difluoro and 2,6-dihalo-3-C1-C6 alkoxy.
Preferred R5 groups include phenyl, 2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl, 2-ethoxyphenyl, 2-methylphenyl, 3-methylphenyl, 2-fluorophenyl, 2,6-difluorophenyl, 2-fluoro-6-methoxyphenyl, 2-fluoro-6-ethoxyphenyl, 2,3,5,6-tetrafluorophenyl, 2-chlorophenyl, 3-chlorophenyl, 1-cyclohexenyl, 2-naphthyl, 2,5-dimethoxyphenyl:, 2-azidophenyl, 2,3,4-trifluorophenyl, 2-fluoro-6-chlorophenyl, 2,6-dimethoxyphenyl, 2,3,6-trichlorophenyl, 2,6-dichlorophenyl, 2,3,5-trichlorophenyl, 3,5-dichlorophenyl, 3-fluorophenyl, 2,4-dimethoxyphenyl, 2-pyridyl, 2-(6-methoxy)pyridyl, 2-(6-ethoxy)pyridyl, 2-(6-fluoro)pyridyl, 2-(5-fluoro)pyridyl, 2-(4-fluoro)pyridyl, 2-(3-fluoro)pyridyl, 2-(6-chloro)pyridyl, 2-(5-chloro)pyridyl, 2-(4-chloro)pyridyl, 2-(3-chloro)-pyridyl, 2-(5-methoxy-6-fluoro)pyridyl, 2-(3-methoxy-6-fluoro)pyridyl, 2-(6-methoxy-3-fluoro)pyridyl, 2-(5-ethoxy-6-fluoro)pyridyl, 2-(3-ethoxy-6-fiuoro)pyridyl, 2-(6-ethoxy-3-fluoro)pyridyl, 2-(5,6-difiuoro)pyridyl, 2-(3,6-difluoro)pyridyl, 2-(5,6-dichloro)-pyridyl, 2-(3,6-dichloro)pyridyl, 2-(6-methoxy)pyridyl, 2-(6-ethoxy)pyridyl, 2-[1,3-pyrimidyl], 2-pyrazinyl, 3-pyridazinyl, 2,6-difluoro-3-methoxyphenyl, 2,6-difluoro-3-ethoxyphenyl, 2,6-difluoro-4-methoxyphenyl, 2,6-difluoro-4-ethoxyphenyl, 2-(3-ethoxy)pyridyl, 2-(3-methoxy)pyridyl, 2,6-difluorophenyl, 2,6-difluoro-3-N-methyl-carboxamidephenyl, 2-fluoro-6-chlorophenyl, 3-bromo-6-methoxyphenyl, 3-ethoxyphenyl, 3-bromo-6-ethoxyphenyl, 3-(2-fluoro)pyridyl, (2-vinyl)phenyl, (3-vinyl)phenyl, (3-methoxy-carbonyl)phenyl, 5,6-dimethylbenzotriazolyl, 2,3-difluoro-6-methoxyphenyl, 2,6-difluoro-3-cyanophenyl, 3-ethynylphenyl, and 2,5-diethoxyphenyl.
A further aspect of the invention provides novel compounds within the above Formula 1A, wherein A is as defined above;
R1 is a stable, unsaturated, substituted or unsubstituted heterocycle having i) a 3 to 8 membered monocyclic ring with up to 4 hetero atoms or ii) a 7 to 10 membered bicyclic ring with up to 5 hetero atoms wherein the Nxe2x80x2-bonded ring has at least one of the hetero atoms and wherein the hetero atoms are selected from N, O and S;
R2 is a group of the formula 
xe2x80x83wherein R5 is a stable, saturated or unsaturated, substituted or unsubstituted, i) 3 to 8 membered organic monocyclic ring having 0 to 4 hetero atoms or ii) a 7 to 10 membered organic bicyclic ring having 0 to 5 hetero atoms, the hetero atoms being selected from S, O and N;
and R6, R7, R8 and R9 are independently C3-C8 cycloalkyl, hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted C1-C6 alkyl, substituted C2-C6 alkenyl, or substituted C2-C6 alkynyl, C1-C6 substituted alkoxy, halo, amino, nitro, cyano, C1-C5 alkoxy, hydroxy, hydroxymethyl, aminomethyl, carboxymethyl, C1-C4 alkylthio, C1-C4 alkanoyloxy, carbamoyl, or a halo substituted C1-C6 alkyl; or two of which, along with the carbons to which they are attached, combine to form a stable, saturated or unsaturated, substituted or unsubstituted, 3 to 7 membered organic monocylic ring having 0 to 4 hetero atoms selected from S, O, or N; or R6 and R8, or R7 and R9, along with the carbon to which they are attached, form a stable, saturated or unsaturated, substituted or unsubstituted, 3 to 7 membered organic monocylic ring having 0 to 4 hetero atoms selected from S, O, or N; or a pharmaceutically acceptable salt thereof, with the provisos that:
a) if R6-R9 are all hydrogen, then the following combinations are excluded:
b) if R7 is methyl or together with R6 forms cyclopropyl and the remainder of R6-R9, are hydrogen, then the following combination is excluded:
c) if R7 is alkylthio, and the remainder of R6-R9 are hydrogen, then the following combination is excluded
The definitions above also apply to these compound aspects of the invention with She exception that aryl and substituted aryl are excluded as possible optional substituents to group R1.
Further, preferred compounds of the invention are those in which R1, and R6-R9 are as defined above and R5 is substituted or unsubstituted C2-C6 alkenyl, C2-C6 alkynyl or wherein R5 is xe2x80x94OR10 or xe2x80x94C(O)R10 where R10 is R5 as defined above, or substituted or unsubstituted C2-C6 alkenyl or C2-C6 alkynyl.
It is believed that none of the compounds excluded by the provisos except proviso x) have been known in a therapeutic role and thus, a further aspect of the invention includes the compounds embraced by the provisos for use in therapy.
As mentioned above, the invention includes pharmaceutically acceptable salts of the compounds defined by the above formula (I). Although generally neutral, a particular compound of this invention can possess a sufficiently acidic, a sufficiently basic, or both, functional groups, and accordingly react with any of a number of nontoxic inorganic bases, and nontoxic inorganic and organic acids, to form a pharmaceutically acceptable salt. Acids commonly employed to form acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid and the like, and organic acids such as p-toluene sulfonic, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like. Examples of such pharmaceutically acceptable salts thus are the sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, g-hydroxybutyrate, glycollate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate and the like. Preferred pharmaceutically acceptable acid addition salts are those formed with mineral acids such as hydrochloric acid and hydrobromic acid, and those formed with organic acids such as maleic acid and methanesulfonic acid.
Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and One like. Such bases useful in preparing the salts of this invention thus include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium bicarbonate, calcium hydroxide, calcium carbonate, and the like.
The pharmaceutically acceptable salts of the invention are typically formed by reacting a compound as defined with an equimolar or excess amount of acid or base. The reactants are generally combined in a mutual solvent such as diethyl ether or benzene, for acid addition salts, or water or alcohols for base addition salts, and the salts normally precipitate out of solution within about one hour to about ten days and can be isolated by filtration or other conventional methods. The salts of the compounds of the invention will convert to the compound Per se after administration and are thus prodrugs. All prodrugs are administered in an amount sufficient to generate an effective amount of the compound to contact the virus and interact with it (e.g. inhibit replication thereof).
The compounds of the present invention also include racemates, racemic mixtures, and individual enantiomers or diastereomers. All asymmetric forms, individual isomers and combinations thereof are within the scope of the present invention.
As noted, the optically active diastereomers of the compounds of Formula 1 are considered part of this invention and such optically active isomers may be prepared from their respective optically active precursors by the procedures described herein, or by resolving the racemic mixtures. The resolution can be carried out in the presence of a resolving agent, by chromatography, by repeated crystallization or by some combination of these techniques which are known to those skilled in the art. Further details regarding resolutions can be found in Jacques, et al., Enantiomers, Racemates, and Resolutions, John Wiley and Sons 1981.
The compounds of the present Invention, or their precursors, are prepared using procedures known to those of ordinary skill in the art. More particularly, the compounds of Formula (1) are prepared according to the general procedures shown below in Schemes I-IV, and VII, as described below.
In Scheme I, a derivative of an isocyanate 
or isoselenocyanate 
is reacted with with an amino group (2) in approximately 1:1 molar ratio, in an inert organic solvent, such as N,N-dimethyl formamide and stirred at an appropriate temperature of between about 0-150xc2x0 C. for a period of time between 1 and 72 hours. The time and temperature used depends upon he reactivity of the individual reagents. The product (3) may be isolated by conventional techniques.
Scheme II is run under the same general reaction conditions as Scheme I. Where 
Schemes I and II are processes analogous to that described in Advanced org. Chem. 3rd Ed., J. March, p 802, 1985, J. Wiley and Sons and references cited therein, incorporated herein by reference. Wherein 
Schemes I and II are processes analogous to that described in Ber. Deutsch Chem. Ges., 100, 1459, 1967; J. Chem. Soc. Chem. Commun., 372, 1968; J. Prakt. Chem., 315, 155, 1973 and references cited therein, incorporated herein by reference. Wherein A=SO2, Schemes I and II are processes analogous to that described in J. Am. Chem. Soc., 94, 6135,.1972 and Org. 
Prep. Proced. Int., 16, 49, 1984 and references cited therein, incorporated herein by reference. 
In Scheme III, a derivative of an amine (7) and an amine (8) are added in an inert solvent, such as N,N-dimethyl formamide, tetrahydrofuran, ethyl acetate, or benzene, to an appropriately substituted derivative (9) wherein L1 and L2 represent leaving groups. The reaction is stirred at an appropriate temperature of between about 0-150xc2x0 C. for a period of time between 1 and 72 hours. The time, temperature, and sequence of addition used depends upon the reactivity of the individual reagents. The product (10) may be isolated by conventional techniques.
Wherein 
Scheme III is a process analogous to that described in J. Org. Chem., 56, 891, 1991 and references cited therein, incorporated herein by reference.
Wherein 
Scheme III is a process analogous to that described in Bull. Chem. Soc. Jpn., 62, 2419, 1989 and references cited therein, incorporated herein by reference.
Wherein 
Scheme III is a process analogous to that described in J. Org. Chem., 24 , 1157, 1959; J. Org. Chem., 38, 155, 1973; J. Med. Chem., 20, 901, 1977; Tetrahedron Lett., 30, 7313, 1989; Org. Prep. Proced. Int., 17, 256, 1985; J. Heterocyclic Chem., 24, 275, 1987; Org. Prep. Proced. Int., 23, 721, 1991 and references cited herein, incorporated herein by reference.
Wherein 
Scheme III is a process analogous to that described in Angew. Chem. Int. Ed., 26, 1165, 1987; Liebigs Ann. Chem., 207, 1990; European Patent 58492, 1982; Syn. Commun., 19, 943, 1989; J. Heterocyclic Chem., 26, 1335, 1989; Belgium Patent 894093, 1982; Syn. Commun., 21, 1213, 1991; Synthesis, 195, 1990; U.S. Pat. No. 4,968,808; j. Org. Chem., 32, 2661, 1967; Synthesis, 76, 1987 and references cited therein, incorporated herein by reference. Wherein A=SO2, Scheme III is a process analogous to that described in Synthesis, 192, 1983; J. Org. Chem., 45, 5371, 5373, 1980; Org. Syn. Coll. Vol. VI, 78, 1988; J. Prakt. Chem., 29, 328, 1965; Arch. Pharm., 321, 375, 1988; J. Chem. Soc., 4367, 1960; Synthesis, 576, 1972 and references cited therein, incorporated herein by reference. 
In Scheme IV, a thiourea derivative (11) is converted to the corresponding derivative (10). Wherein 
Scheme IV is a process analogous to that describe in J. Org. Chem., 26, 2925, 1961 and Tetrahedron Lett., 27, 3911, 1986 and references cited therein, incorporated herein by reference. Wherein 
Scheme IV is a process analogous to that described in Eur. J. Med. Chem., 16, 317, 1981 and references cited therein, incorporated herein by reference. Wherein 
Scheme IV is a process analogous to that described in J. Org. Chem., 49, 4123, 1984; J. Med. Chem., 20, 901, 1977; Tetrahedron Lett., 30, 7313; 1989; Syn. Commun., 13, 67, 1983; and Curr. Sci., 37, 645, 1968 and references cited therein, incorporated herein by reference. 
In Scheme V, a carbodiimide derivative (12) is reacted with a nucleophile to form the corresponding product (13). Wherein 
scheme V is a process analogous to that described in U.S. Pat. No. 4,414,211, 1980; J. Chem. Soc. C, 1429, 1970; J. Chem. Soc. P. T. 1, 1241, 1977; J. Med. Chem., 32, 228, 1989 and references cited therein, incorporated herein by reference. Wherein 
Scheme V is a process analogous to that described in Monat. fur Chem., 97, 695, 1966 and Chem. Ztq., 112, 107, 1988 and references cited therein, incorporated herein by reference. 
In Schemes VI and VII, derivatives 14 and 15, wherein L represents a leaving group, is reacted with a nucleophile to afford the corresponding derivatives 16 and 17, respectively. Wherein 
Schemes VI and VII are processes analogous to that described in Org. Syn. Coll. Vol. V, 966, 1973 and references cited therein, incorporated herein by reference. Wherein 
Schemes VI and VII are processes analogous to that described in J. Org. Chem., 51, 1882, 1986; Syn. Comm., 20, 217, 1990, Tetrahedron Lett., 1879, 1970, Org. Prep. Proced. int., 17, 256, 1985; J. Heterocyclic Chem., 24, 275, 1987; Org. Prep. Proced. Int., 23, 721, 1991 and references cited therein, incorporated herein by reference. Wherein 
Scheme VI and VII is a process analogous to that described in Ann. Chem., 2096, 1979 and references cited therein, incorporated herein by reference. 
Compounds wherein Zii is S(xe2x95x90O)2 can be prepared by reacting an amine of the formula R2NH2 with diaminosulfone, for instance under reflux in 1,2-dimethoxymethane, to form a compound of the formula R2NHS(xe2x95x90O)2NH2. This, in turn, is subjected to reaction with a compound of the formula H2NR1, for instance, in DMF or 1,2-dimethoxyethane, to form R2NHS(xe2x95x90O)2NHR1. This process is, thus, analogous to that described in Arya et al., Ind. J. Chem. B, 1976, 14B, 766.
Alternatively, compounds wherein Zii is sulphone can be prepared by reacting an amine of the formula R2NH2 with a dihalosulphone, e.g. SO2Cl2, for instance under reflux with SbCl5 and acetonitrile to produce a compound of the formula R2NHS(xe2x95x90O)2halo which can be reacted with the appropriate R1 amine, for example 2-amino, 6-chloropyridyl in acetonitrile and triethylamine. This process is analogous to Liebigs Ann. Chem. 729 (1969) 40.
Compounds wherein Zii is So can be prepared by reducing compounds prepared by the above two methods using conventional reduction techniques. Alternatively, a sulfinyl compound can be prepared by the methodology in Liebigs Ann. Chem. 1979, 1756, where SOCl2 is reacted with several molar equivalents of an aryl leaving group such as pyridyl to form a dipyrid-1-ylsulphinyl intermediate. This, in turn, can be transaminated with appropriate R1 and R2 amines as described in Tetrah. Lett. 1985, 26, 3821, to produce a compound of the structure R2NHSONHR1.
Tests with the above compounds of Formula 1 have indicated activity as inhibitors of HIV. While not being bound by theory, it is believed that the compounds act as reverse transcriptase inhibitors, and thereby act to inhibit replication of the virus.
The HIV inhibition activity of the present invention is generally between 1 and 3 logs greater against HIV-1 strains than against HIV-2 strains. This enables the compounds and compositions of the present invention to be used to selectively inhibit HIV-1 growth in cell cultures where co-infection with HIV-1 and HIV-2 is suspected. Suppression of one strain or the other is necessary to enable appropriate choice of antiviral agent to combat each strain. In practice, selective inhibition of HIV-1 in a co-infected cell culture entails administering a concentration of a compound of the present invention which is intermediate the respective IC50 or more preferably the IC90 for that compound against HIV-1 and HIV-2. The cell culture is subject to an appropriate incubation time, which is selected to be sufficiently long to disable the infectivity of the HIV-1 virions, but not sufficiently long as to encourage the development of resistant mutants, for example between 24 and 48 hours. The supernatent is subject to limiting dilution, preferably in a conventional resuscitation buffer to sequester any remaining compound, until it is statistically likely that only a single infectious dose of HIV-2 virons is left. This is then reinoculated in order to determine, for example, the resistance profile or anti-viral susceptibility of the HIV-2 strain uninfluenced by the co-infection with HIV-1.
The following is a description of the test systems used in analyzing compounds in effectiveness against HIV.
MT4 cells in a medium of RPMI 1640, 5% FCS, penicillin/streptomycin are adjusted to 2xc3x97105 cells/ml and seeded into microplates (96 wells/plate) 100 ml cell suspension/well giving 2xc3x97104 cells/well. The compound to be tested is made into a 10 mg/ml mixture in DMSO and stored at xe2x88x9220xc2x0 C. The compound in DMSO is diluted with medium containing 10% DMSO in a 10-fold dilution series to give 1 mg/ml, 10 mg/ml, and 100 mg/mn solutions. Further dilutions to 400, 40, 4 and 0.4 mg/ml are made in medium containing microplates. Fifty ml of the 400, 40, and 4 mg/ml are transferred to the cell-containing microplates with a multi-channel pipette (final concentration: 100, 10, and 1 mg/ml). Finally, 50 ml of virus suspension is added to each well (with a repetitive xe2x80x9cEppendorfxe2x80x9d multipipett). Each plate has at least six wells with the following: [Test A: HIV virus; Test B: HIV(II) virus; Test C: SIV virus; Test D: No virus]; with no drug (virus control) and six wells without virus (medium control). The plate is put into a plastic bag and incubated for six days in CO2 atmosphere. To each well in the plate is added 50 ml of XTT ((2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-5-[(phenylamin o)carbonyl]-2H-tetrazolium hydroxide), (1 mg/ml 0.01-0.02 mM N-metyl-phenazonium methosulfate). After six hours of incubation in CO2 atmosphere the plates are covered with adhesive plate sealers and gently mixed on a vortex. Optical densities are determined at a wavelength of 450 nm and a reference wavelength of 650 nm. The percent reduction of cytotoxocity caused by the virus infection is calculated as follows:                               OD          450                ⁢                  xe2x80x83                ⁢        compound            -                        OD          450                ⁢                  xe2x80x83                ⁢        inf        ⁢                  xe2x80x83                ⁢        cells                                      OD          450                ⁢                  xe2x80x83                ⁢        uninf        ⁢                  xe2x80x83                ⁢        cells            -                        OD          450                ⁢                  xe2x80x83                ⁢        inf        ⁢                  xe2x80x83                ⁢        cells              xc3x97  100
MT-4/H9-cells are adjusted to 2xc3x97105 cels/ml medium (RPMI 1640, 5% FCS, penicillin/streptomycin) and seeded into microplates (96 wells/plate) 100 ml cell suspension/well giving 2xc3x97104 cells/well. The compound to be tested is made 10 mg/ml in DMSO=stock solution (stored at xe2x88x9220xc2x0 C.). The compound dissolved in DMSO is diluted 25 times in medium to give 400 mg/ml. Further dilutions to 40 mg/ml and 4 mg/ml are made in microplates.
50 ml of the dilutions 400 mg/ml, 40 mg/ml and 4 mg/ml are transferred to the xe2x80x9ccell-containingxe2x80x9d microplate with a multichannel pipette. (Final concentration: 100, 10 and 1 mg/ml).
Finally 50 ml of virus suspension is added to each well (with a repetitive xe2x80x9cEppendorf multipettxe2x80x9d). [Test E-HIV-1; Test F-HIV-2; Test G-SIV; Test H-no virus].
Each plate has at least four wells with virus but no drug (virus control) and two wells without virus (medium control). The plate is put into a plastic bag to avoid evaporation and incubated for six days in CO2-atmosphere. 10 ml supernatant from each well is transferred with a multichannel pipette into a new microplate to which 40 ml VDB, (50 mM Tris-HCl pH=7.6, 35 mM KCl, 4 mM DTT, 1 mM EDTA, 1.3% Triton X-100), have been added to each well. The addition of 50 ml RT-reaction mix, (10 ml culture supernatant, 40 ml VDB and 50 ml reaction mixture giving a final concentration of: 100 mM Tris-HCl pH=7.6, 100 mM KCl, 4 mM MgCl2, 4 mM DTT, 275 mg/ml BSA/ml, 5 mg (rA)n(dT)12-18/ml and 0.3 mM 3H dTTP (specific activity 18.000 cpm/pmol)) gives a final volume of 100 ml/well. After 60 minutes of incubation the whole assay volume is transferred by use of a cell harvester to a filter mat prewetted with 5% TCA. The filter is washed in 5% TCA and rinsed once in ethanol. After drying the filter mat at 60xc2x0 C. for 30 min. each filter (96/mat) is punched out and put into counting vials 2 ml of scintillation fluid is added and the samples are counted (1 min) or the whole filter mat is put into a plastic bag, 10 ml of scintillation fluid is added and the filter mat is counted in a Beckman Betaplare counter. Percent reduction of RT activity is determined by comparing RT activity for virus control with the RT activity measured for each dilution of the compound.
The compounds were tested for direct inhibitory activity on HIV-RT in a volume of 100 ml recombinant HIV-RT (diluted in virus disruption buffer to give 200.000 cpm)
100 MM Tris-HCl pH 7.6, 100 mM KCl, 4 mM MgCl2, 4 mM DTT, 275 mg/ml BSA, 0.5 mg (rA)n(dT)12-18 and 0.3 mM 3H-=dTTP (specific activity 18.000 cpm/mol). After 60 minutes of incubation 40 ml in duplicate were spotted on paper discs and washed in 5% TCA. After rinsing the paper discs in ethanol they were dried and counted in scintillation fluid.
The following illustrate activities of compounds in the above-described tests. The numbers represent % inhibition.
A feature of this invention also disclosed is a method of administering to a human in need thereof the compounds described, their pharmaceutically acceptable salts or pro-drugs thereof to treat or inhibit HIV/AIDS, to inhibit the replication of the HIV/AIDS virus in infected human cells and to inhibit AIDS from developing n humans infected with the HIV/AIDS virus or carrying antibodies to the HIV/AIDS virus.
The present invention also discloses the compounds of the invention and their salts for use in the treatment of the condition referred to above, as well as the use of such compounds in the preparation of pharmaceutical formulations for the treatment of such conditions.
In general for the treatment as described above, a suitable effective dose of the compound or its pharmaceutically acceptable salt will be in the range or 0.5 to 250 mg per kilogram bodyweight of recipient per day. Administration may be by any suitable route including oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. It will be appreciated that the preferred route may vary with, for example, the condition, age, and weight of the recipient.
The administered ingredients may be used as a therapy in conjunction with other therapeutic agents, (other anti-virals, anti-bacterials, compounds useful for preventing resulting secondary or contemporaneous afflictions associated with HIV/AIDS) such as AZT, ddI, ddC, 9-[[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl]guanine, 9-(2-hydroxyethoxymethyl)guanine(acyclovir), 2-amino-9-(2-hydroxyethoxymethyl)purine, suramin, ribavarin, antimoniotungstate (HPA-23), interferon, e.g., a interferon, interleukin II, and phosphonoformate (Foscarnet) or in conjunction with other immune modulators including bone marrow or lymphocyte transplants or other medications such as levamisol or thymosin which would increase lymphocyte numbers and/or function as is appropriate.
While it is possible for the administered ingredients to be administered alone, it is preferable to present them as part of a pharmaceutical formulation. The formulations of the present invention comprise at least one administered ingredient, as above-defined together with one or more acceptable carriers thereof and optionally other therapeutic ingredients. The carrier(s) must be xe2x80x9cacceptablexe2x80x9d in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
The formulations include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. The formulations may conveniently be presented in unit dosage form, e.g., tablets and sustained release capsules, and may be prepared by any methods well known in the art of pharmacy.
Such methods include the step of bringing into association the to be administered ingredients with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.
Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of he active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion and as a bolus, etc.
With regard to compositions for oral administration (e.g. tablets and capsules), the term xe2x80x9csuitable vehiclexe2x80x9d means common excipients such as binding agents, for example, syrup, acacia, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone (Povidone), methylcellulose, ethylcellulose, sodium carboxy-methylcellulose, hydroxypropylmethylcellulose, sucrose and starch; fillers and carriers, for example corn starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid; disintegrators such as microcrystalline cellulose, corn starch, sodium starch glycolate, alginic acid; and lubricants such as magnesium stearate and other metallic stearates, stearic acid, silicone fluid, talc, waxes, oils and colloidal silica. Flavoring agents such as peppermint, oil of wintergreen, cherry flavoring or the like can also be used. It may be desirable to add a coloring agent to make the dosage form more aesthetically pleasing in appearance or to help identify the product. The tablets may also be coated by methods well known in the art.
A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, preservative, surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein.
Formulations suitable for topical administration include lozenges comprising the ingredients in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising he ingredient to be administered in a suitable liquid carrier.
Formulations suitable for topical administration to the skin may be presented as ointments, creams, gels and pastes comprising the ingredient to be administered and a pharmaceutically acceptable carrier. An exemplary topical delivery system is a transdermal patch containing the ingredient to be administered.
Formulations for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.
Formulations suitable for nasal administration wherein the carrier is a solid include a coarse powder having a particle size, for example, in the range 20 to 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations wherein the carrier is a liquid, for administration, as for example, a nasal spray or as nasal drops, include aqueous or oily solutions of the active ingredient.
Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.
Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, or example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets of the kind previously described.
Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose, as herein above recited, or an appropriate fraction thereof, of the administered ingredient.
The antiviral compounds of Formula I can be used as surface disinfectants. Solutions containing as little as 0.1 percent by weight of the antiviral compound maybe effective for disinfecting purposes. Preferably, such solutions also can contain a detergent or other cleansing agent. The solutions maybe useful for disinfecting objects such as glassware, dental and surgical instruments, and surfaces such as walls, floors, and tables in areas where maintenance of sterile conditions is important, for example, hospitals, food-preparation areas, and the like.
In practicing the method for treating or inhibiting HIV and/or AIDS, the antiviral can be administered in a single daily dose or in multiple doses per day. The treatment regime may require administration over extended periods of time, e.g., for several days or for several months or years. The amount administered per dose or the total amount administered will depend on such factors as the nature and severity of the infection, the age and general health of the patient, the tolerance of both the patient and the microorganism or microorganisms involved in the infection to the antiviral compound.
The following formulation examples represent specific pharmaceutical formulations employing compounds comprehended by the present method. The formulations may employ as active compounds any of the compounds of Formula I or a pharmaceutically acceptable salt thereof. The examples are illustrative only and are not intended to limit the scope of the invention in any way.
Hard gelatin capsules are prepared using he following ingredients:
The above ingredients are mixed and filled into hard gelatin capsules in 460 mg quantities.
A tablet formula is prepared using the ingredients below:
The components are blended and compressed to form tablets each weighing 675 mg.
An aerosol solution is prepared containing the following components:
The active compound is mixed with ethanol and the mixture added to a portion of the propellant 22, cooled to xe2x88x9230xc2x0 C. and transferred to a filling device. The required amount is then placed in a stainless steel container and diluted with the remainder of the propellant. The valve units are then fitted to the container.
Tablets each containing 60 mg of active ingredient are made up as follows:
The active ingredient, starch and cellulose are passed through a No. 45 mesh U.S. sieve and mixed thoroughly. The solution of polyvinylpyrrolidone is mixed with the resultant powders which are then passed through a No. 14 mesh U.S. sieve. The granules so produced are dried at 40-60xc2x0 C. and passed through a No. 18 mesh U.S. sieve. The sodium carboxymethyl starch, magnesium stearate and talc, previously passed through a No. 60 mesh U.S. sieve, are then added to the granules which, after mixing, are compressed on a tablet machine to yield tablets each weighing 150 mg.
Capsules each containing 80 mg of medicament are made as follows:
The active ingredient, cellulose, starch and magnesium stearate are blended, passed through a No. 45 mesh U.S. sieve, and filled into hard gelatin capsules in 200 mg quantities.
Suppositories each containing 225 mg of medicament are made as follows:
The active ingredient is passed through a No. 60 mesh U.S. sieve and suspended in the saturated fatty acid glycerides previously melted using the minimum heat necessary. The mixture is then poured into a suppository mold of nominal 2 g capacity and allowed to cool.
An intravenous formulation is prepared as follows:
The solution of the above ingredients is administered intravenously at a rate of 1 ml/minute to a mammal in need of treatment.
It should be understood that in addition to the ingredients particularly mentioned above the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question.
The compounds of the present invention are also useful as laboratory tools for monitoring mutation of reverse transcriptase in vitro. For example, the compounds of Formula 1A stabilize the reverse transcriptase enzyme in vitro and allow for convenient cocrystallization. In its native state, reverse transcriptase is difficult to crystallize, a necessary initial step for X-ray crystallographic determination of the spatial structure of the enzyme. Repeated monitoring of the fine structure of this enzyme is necessary due to its rapid mutation in vivo, which in turn, leads to rapid viral resistance against conventional anti-HIV agents such as AZT and ddI. The rapid determination of the mutated structures is a key step to effective multiple therapy and slowing down the development of HIV resistant strains.
The limited success in growing reverse transcriptase crystals by conventional techniques has prompted a search for alternative methodologies and a certain amount of success has been accomplished with triple complexing of HIV reverse transcriptase with Fab fragments and DNA (A. Jacobo Molina et al. Proc. Nat""l. Acad, Sci. USA 90:6320 (1993)). However, the need for rapid and robust preparation of transcriptases for structural determination motivates the search for simpler and more effective cocrystallizing stabilizers.
Accordingly, a further aspect of the invention provides a method for stabilizing HIV-1 reverse transcriptase for crystallization comprising the steps or contacting a, preferably pure, solution of the transcriptase with a stabilizing amount of a compound of Formula 1A and cocrystallizing the resulting complex by conventional techniques such as the equilibrated hanging drop method or vapour diffusion method. Alternatively, ready seeded incipient crystals of transcriptase can be stabilized by conventional soak techniques to produce crystals having superior regularity and a longer working life.
Preferably, the compound of Formula 1A is present in a slight molar excess, for example a twofold molar excess, over the concentration of the transcriptase, however the concentration of the compound of the invention can vary within broad margins, for example between 1:10 (stabilizer:transcriptase) to 10:1.
Conventional crystallizing buffers, pH and osmolality regulators, etc. may be used in the mother liquor as are exemplified in A. J. McPherson et al., Methods Biochem. Anal. 23:249-345 (1979).
A crystallization buffer comprising 14% PEG, 0.1M Tris, pH 8.5, 0.36 mM HIV-1 reverse transcriptase (216 fragment) and 0.4 mM of the compound N-(2-imidazo-4-yl)ethyl)-Nxe2x80x2-(5-bromopyrid-2-yl)-urea (Example 36) is subject to hanging drop crystallization (T. Unge et al. Aids Res. and Human Retroviruses 6:1297 (1990). The resulting crystals are significantly larger (elongate prisms 2-4 mm long) and macroscopically more regular than those crystallized without the compound of the present invention. Preliminary results indicate that the useful exposure life is in excess of 10 hours, making the crystals amenable to X-ray crystallography to at least 3.5 xc3x85, a threefold improvement.
Alternatively, co-crystallization can be effected by vapour diffusion, wherein a purified HIV-1 reverse transcriptase is prepared, e.a., in a crystallizing buffer consisting of 25 mM bis tris propane, pH 7, 50 mM ammonium sulphate, 0.1% (w/v) xcex2-octyl glucoside, 5% (v/v) polyethylene glycol 8 000 and 0.1% sodium azide. A twofold molar excess of the compound of the invention over the concentration of transcriptase is added and allowed to complex. Crystals are crown by vapour diffusion against a solution which is double in the concentration of all components except the transcriptase. Preliminary results suggest that the resulting crystals can have dimensions in excess of 3.0xc3x970.6xc3x970.4 mm and a useful exposure lifetime of greater than 10 hours, enabling X-ray crystallography down to 3.5 xc3x85. This method should be contrasted against crystals grown without co-crystallization which are irregular and only amenable to X-ray crystallography to 9.4 xc3x85 resolution (T. Unge et al. Aids Res. and Human Retroviruses 6:1297 (1990)).
The following examples further illustrate the compounds of the present invention and methods for the synthesis. The examples are not intended to be limiting to the scope of the invention in any respect and should not be so construed. In the descriptions of the preparations, the reference to example numbers are those found in Patent Application WO 93/03022 having a publication date of February 13, 1993, hereby incorporated by reference.
The title compound was obtained starting from 2,6-difluorobenzaldehyde (purchased from Aldrich) which was converted to cis-2,6-difluorophenylcyclopropylamine according to the procedure in Example 375 and then condensed with the product from Example 393 according to the procedure in Example 370; 1H-NMR (250 MHz, DNSO-D6). d 1.3-1.4 (q, 1H) 1.6-1.7 (q, 1H) 2.3-2.4 (q, 1H), 3.8-3.9 (m, 1H) 7.2-7.3 (m,3H) 7.4-7.5 (m,1H) 7.7 (d, 1H) 7.8-7.9 (m, 1H) 10.7010.8 (s,1H) 11.0-11.1 (d, 1H).
The starting material (xc2x1)-cis-2-(2-chloro-3-ethoxy-6-fluorophenyl)cyclopropylamine was prepared from 2-chloro-4-fluorophenol in a manner analogous to Examples 362, 375 and 348. 5-Chloropyrid-2-ylisothiocyanate was prepared as in Example 374 and then condensed with (xc2x1)-cis-2-(2-chloro-3-ethoxy-6-fluorophenyl)cyclopropylamine in a manner analogous to Example 370 to give the titled product.
1H-NMR (250 MHz, CDCl3) d 11.25 (br s, 1H), 9.09 (br s, 1H), 7.72 (d, 1H), 7.49 (dd, 1H), 6.94 (t, 1H), 6.84 (dd, 1H), 6.74 (d, 1H) 4.12 (q, 2H), 3.67-3.57 9 (m, 1H), 2.23 (q, 1H), 1.78-1.68 (m, 1H), 1.55 (t, 3H), 1.44-1.36 (m, 1H). 13C-NMR (62.9 MHz, CDCL3) d 180.6, 156.6 (d), 114.2 (d), 112.8, 118.8 (d), 65.0, 32.7, 16.5, 15.7 (d), 14.7. Anal. calcd. for C17H16Cl2FN3OS: C, 51.01; H, 4.03; N, 10.5. Found C, 50.9; H, 3.9; N, 10.25.
The starting material 2-chloro-3-ethoxy-6-fluorophenethylamine was prepared from 2-chloro-3-ethoxy-6-fluorobenzaldehyde in a manner analogous to Example 151 and 2-chloro-3-ethoxy-6-fluorobenzaldehyde was prepared from 2-chloro-4-fluorophenol in a manner analogous to Example 362. 2-Chloro-3-ethoxy-6-fluorophenethylamnine was condensed with the product of Example 392, using the procedure of Example 411 to give the titled product.
1H-NMR (250 MHz, CDCl3): d 11.30 (br s, 1H, NH), 9.35 (s, 1H, NH), 8.08 (d, 1H, pyridine), 7.69 (dd, 1H, pyridine) 6.93-6.76 (m, 3H, phenyl, pyridine), 4.11-4.00 (m, 4H CH2xe2x80x94NH, OCH2CH3), 3.24 (t, 2H, phenyl-CH2), 1.46 (t, 3H, OCH2CH3). 13C-NMR (250 MHz, CHCl3): 179, 157, 153, 151, 150, 146, 140, 125, 124, 113, 112, 111, 65, 44, 25, 14. Anal. Calcd, for C16H16BrC1FN3OS: C, 44.4; H, 3.7; N, 9.7 Found: C, 44.7; H, 3.9; N, 9.3.